The emergence of a novel human coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected in Wuhan, China, in December 2019. SARS-CoV-2 causes the coronavirus disease 2019 (COVID-19), a heterogeneous disease. Due to its rapid spread across the world within a short period, by March 2020, the World Health Organization (WHO) declared COVID-19 a global pandemic. To date, SARS-CoV-2 has infected over 126.4 million lives and caused over 2.7 million deaths globally.

Study: Accelerated RNA detection using tandem CRISPR nucleases. Image Credit: Orpheus FX / Shutterstock

Many approaches have been adopted to mitigate SARS-CoV-2 infection and manage COVID-19’s spread. Strategic non-pharmaceutical approaches have been in place in many parts of the world, including lockdowns, social distancing, physical protective wear such as masks and other gear, and travel-restrictions. Even as approved vaccines against the SARS-CoV-2 are currently administered across the world, the threat from the SARS-CoV-2 and future zoonotic viruses still exist.

An essential component of pandemic control and future prevention is extensive testing at the point-of-care. This enables enhanced detection, making subsequent isolation measures and healthcare access for the infected individuals possible.

The qRT-PCR (quantitative reverse-transcriptase-polymerase chain reaction), employed for RNA detection in clinical samples, provides high sensitivity; the limit of detection is ~1 molecule/µl. However, this technique is too complex to implement for rapid point-of-care testing.

In an alternative approach to the PCR-based methods, CRISPR-Cas proteins have been employed by a large team of researchers to identify the SARS-CoV-2 genomic RNA in patient samples with PCR-derived cycle threshold (Ct) values up to 22 – corresponding to ~1600 copies/µl in the assay. This study was recently posted on the medRxiv* preprint server.

Here, the researchers showed that unrelated CRISPR nucleases could be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ∼30 RNA copies/microliter in 20 minutes. Thus, the assay – Fast Integrated Nuclease Detection In Tandem (FIND-IT) – combines RNA-guided Cas13 and Csm6 with a chemically stabilized activator and creates a one-step RNA detection for effective use at point-of-care.

CRISPR-Cas proteins are simple, programmable, RNA-guided recognition of RNA sequences that trigger cleavage and release of a fluorescent reporter molecule, which can be used to detect any RNA sequence. However, long reaction times hamper sensitivity and speed of the reaction; these are disadvantages for a point-of-care testing application. This study addressed the challenges and optimized the reaction to achieve the best results.

They reported an amplification-free RNA detection method using L. buccalis Cas13 (LbuCas13) with three guide RNAs, to rapidly identify the SARS-CoV-2.

On a mobile phone-based imaging device, the researchers showed that this assay detects as low as 200 cp/µl of target RNA within 30 minutes. However, they noted that increased speed and sensitivity of the one-pot detection chemistries are still needed to enable their widespread use for point-of-care diagnostics.

In addition to sensitivity and speed, to meet the accuracy threshold set by the Food and Drug Administration (FDA) for emergency use authorization, the researchers analyzed this assay in replicates. They established that the assay met the 95% accuracy requirement for 20 replicates at 31 cp/µl of viral RNA in 60 min (the limit of detection as defined by the FDA).

The researchers also developed a detector consisting of a microfluidic chip with reaction chambers, a heating module that maintains the reactions at 37˚C, and a compact fluorescence imaging system. This enables ease of use at the point-of-care.

They also demonstrated that the FIND-IT assay could detect SARS-CoV-2 genomic sequences in total RNA extracted from nasopharyngeal patient samples, with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system.

In this study, the researchers have optimized and standardized the detection to a time frame better suited to point-of-care testing. They established that the tandem CRISPR nuclease assay could detect RNA sequences from an infectious pathogen rapidly and with high sensitivity.

This study demonstrated the value of FIND-IT to stabilize the Csm6 nuclease activation and enable its tandem use with RNA-cleaving CRISPR-Cas proteins for direct and rapid RNA detection.

The researchers noted that the FIND-IT is a one-step assay that enables rapid RNA detection with sensitivity, accuracy, and adaptability suitable to a point-of-care detection of virtually any RNA sequence.

This technology could enable practical on-site detection of viral or human RNA in clinical samples, or plant, fungal, or microbial RNA in environmental samples.”

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Ramya Dwivedi

Ramya has a Ph.D. in Biotechnology from the National Chemical Laboratories (CSIR-NCL), in Pune. Her work consisted of functionalizing nanoparticles with different molecules of biological interest, studying the reaction system and establishing useful applications.